JP2001526538A - Uncoupling protein homolog: UCP3 - Google Patents

Abstract

(57) Abstract The present invention relates to cloned genes encoding uncoupling proteins that regulate thermogenesis in human skeletal muscle and heart. A further aspect of the invention relates to the use of the gene for correcting thermogenic dysfunction in human skeletal muscle and heart. The present invention allows for the use of novel methods of treating (or preventing) diseases such as obesity or cachexia. As a result of the identification and isolation of the genes encoding UCP3 _L and UCP3 _S , the basis for correction of UCP3 deficiency or excess is effectively due to gene therapy or antisense oligonucleotides for the sequence for the gene of interest or one of its fragments. It becomes possible to develop a drug that acts as a drug.

Description

DETAILED DESCRIPTION OF THE INVENTION                     Uncoupling protein homolog: UCP3   The present invention relates to uncoupling proteins that regulate thermogenesis in human skeletal muscle and heart (UCP3_L )).   The present invention also relates to uncoupling proteins (UCP) that regulate thermogenesis in human skeletal muscle and heart. Three_S)).   A further aspect of the present invention is a method for correcting thermogenetic dysfunction in human skeletal muscle and heart. The use of the cloned gene for positive.   Dysfunction of thermogenesis can lead to diseases such as obesity or cachexia.   Obesity is characterized by an excess of fat, which can represent more than 30% of body weight. D The incidence of this disorder of energy balance has been increasing steadily in industrialized countries. Prevention of the onset of obesity, or treatment thereof, is dependent on the pathology, ie, cardiovascular disease, hypertension. And to avoid complications associated with type II diabetes.   In certain embodiments, controlling heat generation has also proven beneficial. For humans Thus, weight loss due to weight loss diets induces body energy savings. Normal Energy consumption depends on the body's previously lost fat mass and lean body mass It stays low until mass regains. This reduction in energy consumption is often Guides return to excess weight. Athletes who had been exercising had similar problems, As soon as you stop running, you come across. Effectively smells trained humans Thus, energy consumption is reduced compared to sedentary people. This energy saving Responsible for substantial weight gain (most notably fat) after cessation of sustained physical activity .   Cachexia is a metabolic condition in which energy expenditure exceeds food intake. Its main Causes include inadequate food intake (e.g., anorexia nervosa), cancer, AIDS and shock Infectious diseases including conditions. Loss of fat and muscle mass threatens individual survival I can do it.   Energy expenditure is increased in mitochondria by uncoupling oxidative phosphorylation . Oxidation is effected by protons (H⁺) Is released, dissipated and ATP Produces a proton gradient that allows for formation. Uncoupling is 2,4-dinitrophenol (DNP) and other acidic aromatic compounds. These substances are H⁺From the outside to the inside of the inner mitochondrial membrane. this In the presence of these uncoupling agents, NADH oxidation proceeds normally, but the proton gradient disappears. ATP is not formed by mitochondrial ATP synthetase because it is dispersed.   The abundant brown adipose tissue (BAT) in mitochondria is dedicated to this process of thermogenesis. Gate. The inner membrane of the mitochondria contains large amounts of uncoupling protein (UCP), This allows protons to return from outside to inside of mitochondria. Essence In, uncoupling proteins, mitochondrial proton battery short circuit Produces heat. Despite tissue specificity, UCP is particularly Induces ATP / ADP, including a and 2-oxoglutarate / malate carrier He is a member of the mitochondrial carrier family. A structural carrier In contrast to ATP / ADP carriers, UCP is subjected to a substantial regulatory process (M. Kling Berg, J .; Bioenerg. Biomembr. 25, 447 (1993)). Its activity is Is reduced by triphosphate nucleotides and increased by fatty acids (J. Nedergaard, B. Cannon, New Comprehensive Biochemistry (Bioenergetics) L. Ernster, Ed (Elsevier Science, Stockholm, 1992), vol. 23, 385).   Uncoupling of oxidative phosphorylation is very useful from a biological point of view; To maintain the physiological temperature of newborn animals (including humans) and mammals in cold regions Is the ability of BAT to generate heat.   The human UCP gene is located at 4q31; it consists of six exons and Encodes a 307 amino acid protein without a resequencing signal. other Like mitochondrial carriers, UCPs consist of six exons each. Inserted into the membrane by six encoded hydrophobic α-helical domains (L.P.K. ozaketal., J. Biol. Chem. 263, 12274 (1988)). The polypeptide chains are each Approximately 100, encoded by two exons and corresponding to two transmembrane domains Consists of three related sequences of tandem amino acids (F. Bouillaud et al., Bioche m. Biophys. Res. Commun. 157, 783 (1988)). Topological experiments were based on amino- and UCP of UCP. And carboxy termini are oriented toward the cytosol side of the inner mitochondrial membrane. (B. Miroux et al., EMBO J. 12, 3739 (1993)). Furthermore, J . Nedergaard et al. (New Comprehensive Biochemistry (Bioenergetics) L.E. rnster, Ed. (Elsevier Science, Stockholm, 1992), vol. 23, 385) and D.I. R icquier et al. (FASEB J. 5, 2237 (1991)) states that the expression of UCP is noradrenaline. This indicates that the transcription level increases. This effect is due to the β-adrenoceptor ー (β₁, Β_TwoAnd β_Three) And α₁-Adrenoceptor stimulation Mediated by   However, M. E. J. Lean et al. (Brown Adipose Tissue P. Trayhurn, D. G. Nicholls, Eds, (Edward Arnold, London, 1986), 339) Shows that UCP-expressing BAT is significantly reduced in neonates compared to adults You. Therefore, under physiological conditions, BAT plays an important part in nontremor thermogenesis in humans Can not do.   On the other hand, L. Simonsen et al. (Int. J. Obes. 17, S47 (1993)) is a human, muscle Up to about 40% has been measured to contribute to adrenaline-induced thermogenesis. Luck Experiments performed in PL Thurlby et al., Can. J. Physiol. Pharmacol. 64, 1 111 (1986); Nagase et al. Clin. Invest. 97, 2898 (1996)) is a human skeleton Adrenaline-induced thermogenesis in muscle (L. Simonsen et al., Int. Obes. 17, S4 7 (1993)), indicating that it is mediated by muscle UCP.   In the search for homologues for human skeletal muscle UCP, we identified human skeletal muscle cDNA The library was screened and three clones (UCP2, UCP3_LAnd UCP3_S) Released. UCP2 mRNA is already available from Fleury et al. (Nature Genet. 15, 269 (1997)) As described, UCP3 has been found in all human tissues, but human skeletal muscle Has proven to be very specific. New members of this UCP3 family Is involved in oxidative phosphorylation in humans with strong specificity of expression in skeletal muscle. You.   UCP3 in terms of nucleotides and amino acids_LAnd UCP3_SThe gene encoding The characterization of was first described herein.   One embodiment of the present invention therefore provides that the fragment comprises the amino acid set forth in SEQ ID NO: 4. Uncoupling protein characterized by sequence (UCP3_L), SEQ ID NO: 3 Or comprising the same amino acid sequence A DNA fragment comprising a coding homology sequence. Like Alternatively, the selected DNA fragment is from human skeletal muscle.   In another embodiment of the present invention, the DNA fragment comprises the amino acid sequence shown in SEQ ID NO: 6. Uncoupled protein (UCP3_S) Encoding the nucleotide shown in SEQ ID NO: 5 A homologous sequence characterized by containing the sequence or encoding the same amino acid sequence. A DNA fragment comprising a sequence. Preferably, the selected DNA The fragments are from human skeletal muscle.   One object of the present invention is characterized in that it comprises the amino acid sequence of SEQ ID NO: 4. Is an uncoupling protein.   Another subject of the present invention is characterized in that it comprises the amino acid sequence of SEQ ID NO: 6. Uncoupling protein.   Further, the present invention relates to one of the nucleotide sequences of SEQ ID NO: 3 or 5, Described to provide recombinant DNA sequences, including one of their homologous sequences .   Another object of the invention is a cloning vector into which one of the DNA sequences has been inserted. A DNA molecule, characterized in that the DNA molecule comprises a DNA molecule. Preferably, the clonin The vector is a plasmid or a phagemid.   Another subject of the present invention comprises a nucleotide as set forth in SEQ ID NO: 3, comprising the vector pBLu escript SK^-Is a recombinant DNA molecule inserted into the recombinant DNA molecule. This recombinant DNA molecule is C, Rockville, Md. 20852 USA (ATCC Accession No. 97999-Deposit Date: April 25, 1997).   Another subject of the invention comprises a nucleotide as set forth in SEQ ID NO: 5 and comprises the vector pBlu escript SK^-Is a recombinant DNA molecule inserted into the recombinant DNA molecule. This recombinant DNA molecule is C, Rockville, Md. 20852 USA (ATCC Accession No. 209000-Deposit Date: April 25, 1997).   Another subject of the present invention is a bacterium, characterized in that it comprises one said recombinant DNA molecule. , Yeasts and mammalian cells. Preferably selected The microorganism is an XL-1 Blue MRF 'bacterium (E. coli).   Another embodiment of the present invention relates to a gene as set forth in SEQ ID NO: 3 or 5, and a suitable pharmaceutical vehicle. A pharmaceutical formulation for correcting UCP3 deficiency by gene therapy, including the body. The The gene is preferably an adenovirus, retrovirus, adeno-associated virus , A vector selected from herpes virus, liposome or DNA plasmid Is included.   Construction of adenovirus vectors and production of recombinant adenovirus are performed using standard methods. (Graham et al., Method in Molecular Biology, 1991, V ol. 7, chap. 11, Murray, E. J., Ed., The Humana Press, Inc., Clifton, NJ ; Zabner et al., Cell, 75, 207-216 (1993); Crystal, et al., Nature Genetics. , 8, 42-51 (1994)).   Retroviral vector construction and recombinant retrovirus production are performed using standard methods. (Miller, AD et al., Meth. Enzymol., 217, 581-599 ( 1993); Blaese et al., Human Gene Therapy, 1, 331-362, (1990)).   Construction of adeno-associated virus vectors and recombinant adeno-associated virus is standard (Muzyczka, Curr. Top. Microbiol. Immunol., 158, 97-129). (1992)).   Construction of herpesvirus vectors and recombinant herpesviruses uses standard methods. (Glorioso et al., Semin. Virol., 3, 265-276 (1992)).   Production of UCP3 gene-containing liposomes can be achieved using standard methods (Kirby et al. al., Biotechnology, 2, 979-984 (1984); Felgner et al., Proc. Natl. Acad. S ci. USA, 84, 7413-7417 (1987); Gregoriadis et al., J. Am. Drug Targeting, 3, 4 67-475 (1966)).   DNA plasmid construction and recombinant DNA plasmids are achieved using standard methods. (Lee et al., Cancer Res., 54, 3325-3328 (1994)). Recombinant retrovirus Vector, recombinant adeno-associated virus vector, recombinant herpesvirus vector -UCP3 gene contained in capsid in liposome and recombinant DNA plasmid In each case, the functionality of the sumids was determined by expression of UCP3 in infected cells and UCP3 protein. Depending on the presence of proteins, Northern blotting (Sambrook, et al., Molecular  Cloning, 1989, Nolan, C., Ed., Cold Spring Harbor Laboratory Press, Col d Spring Harbor, NY) and Western blotting (Sambrook, et. al., Molecular Cloning, 1989, Nolan, C., Ed., Cold Spring Harbor Laborat ory Press, Cold Spring Harbor, NY).   In addition, the subject of the present invention is that UCP3_LAnd UCP3_SFragment of the array To correct excess UCP3, including antisense oligonucleotides for Is a medicine.   The present invention therefore utilizes a novel therapeutic (or prophylactic) method for the above types of diseases. Enable. UCP3_LAnd UCP3_SAs a result of the identification and isolation of the gene encoding In effect, gene therapy or the sequence of a gene or fragment thereof for the gene of interest Antisense oligonucleotides related to one can cause UCP3 deficiency or excess It is possible to develop a medicine that acts as a basis for correction.   Gene therapy impairs the function of UCP3 due to one or more mutations in the UCP3 gene. Instructed in all cases. In this case, the UC of the normal gene using an appropriate vector There is transfer to P3-deleted cells. Preferably, the vector is an adenovirus, a reto Virus, adeno-associated virus, herpes virus, liposome or plus Selected from mid. In this case, the UCP3 gene is under the control of an exogenous promoter. is there. Administration of the antisense oligonucleotide may be dependent on UCP3 levels and / or UC It is particularly indicated if the activity of P3 is in excess.   By means of the present invention, in addition to modifying and / or expressing the activity of endogenous UCP3 It is possible to develop drugs that act on the level.   Interventions for the modification of endogenous UCP3 activity, especially by promoting energy expenditure Weight loss in all types of obesity (reduced fat mass and lean mass) (Maintenance) induction. Skeletal muscle UCP3 mRNA levels are higher in fa / fa obese rats Has been shown to be less than in lean Fa /? Rats (O. Boss et al., J. Bi. ol. Chem. 273, 5 (1988)). The same intervention can also follow a restricted diet or meat Applicable to prevent excessive weight recovery after stopping physical training program . Diet restriction induces a decrease in UCP3 gene expression in skeletal muscle (O. Boss et al., J. Biol. Bio. Chem. 273, 5 (1998)), whose expression remains very low for prolonged periods during refeeding. (DW Gong et al., J. Biol. Chem. 272, 24129 (1997)). Continuous exercise training Has also been reported to induce a decrease in UCP3 gene expression in skeletal muscle. (O. Boss et al., FASEB J 12, 335 (1998)), the same intervention has Improve susceptibility to prevent and treat type II diabetes and to increase blood pressure Could be applied for prevention. The same intervention also applies for muscle mass gain in cachectic conditions did it. For the treatment of heart rhythm deficiencies or disorders due to UCP3 dysfunction Was applicable to the treatment of neuromuscular diseases due to UCP3 dysfunction. UCP3 activator or inhibitor The agent could be administered enterally or parenterally.   Modification of the level of endogenous UCP3 expression is also indicated for the above example; This could be done using activators or inhibitors of UCP3 expression. These substances are Could be administered enterally or parenterally. Candidates that may regulate expression are , An activator or inhibitor of a transcription factor specific to UCP3, or a hormone. Instep Thyroid hormone (T3), glucocorticoid (dexamethasone), β-adrenergic Kinetic receptor agonists (Ro 16-8714), leptin and fatty acids are It has been shown to induce an increase in P gene expression (DW Gong et al., J. Biol. Ch. em. 272, 24129 (1997); Larkin et al., Biochem. Biophys. Res. Com. 240 , 222 (1997); Boss et al., Unpublished observations (1997). Cusin et al., Diabetes , (Submitting, 1988). S. Weigle et al., Diabetes 47, 298 (1998)). Meanwhile, instep Hypothyroidism has been shown to be associated with low levels of UCP3 mRNA in skeletal muscle (D . W. Gong et al. Biol. Chem. 272, 24129 (1997)).   UCP3_LAnd / or UCP3_SConstruction of cells containing the gene is achieved using standard methods (Methods in Molecular Biology, 1991, Vol. 7, chap. 2-5, Murray, E . J. Ed., The Humana Press, Inc., Clifton, NJ; Sambrook, et al., Molecula r Cloning, 1989, Nolan, C., Ed., Cold Spring Harbor Laboratory Press, Co. ld Spring Harbor, NY)). UCP3 gene expression in transfected cells Can be assessed by Northern and Western blots. Culture C_TwoC₁₂ Mouse myoblasts were transformed with human UCP3_L Transfected with cDNA and expressed UCP3 (O. Boss et al., J. Biol. Chem. 273, 5 (1998)). . The functionality of the UCP3 protein is determined by the respiration of isolated cells expressing the UCP3 protein or It can be evaluated by measuring the mitochondrial fraction containing UCP3 protein (Methods in Enzymology, Vol. 10, chap. 14, 86-94 (1967)). Measurement of oxygen consumption Is determined by polarography using a Clark electrode (Methods in Enzymol ogy, Vol. 10, chap. 7, 41-48 (1967)). For measurement of mitochondrial membrane potential It is also possible to assess the activity of UCP3 using cytofluorimetry (Methods in Enzymology, Vol. 260, chap. 29, 406-417, chap. 31, 428-447 (1985)). fluorescence Compounds are used for analysis of changes in mitochondrial membrane potential. Cell fluorescence measurement method With the help of UCP3, human UCP3_L C transfected with cDNA_TwoC₁₂Mouse muscle It shows a decrease in blast cell potential (O. Boss et al., J. Biol. Chem. 273, 5 (1998)). UCP (3) Screening of drugs capable of modifying the activity involves screening for isolated cells containing the UCP3 protein. And / or mitochondrial oxygen consumption and / or mitochondrial membrane potential Achieved by measurement. Drugs that modify the activity of UCP3 protein are then Tested in vivo in an animal model showing UCP3 activity that is not or otherwise deleted You.   Antibodies to UCP3 or UCP3 fragments or derivatives thereof are Developed to allow P3 levels to be measured in biological samples. This These antibodies target substances close to UCP3 to modify UCP3 activity. And act on.   In addition, transgenic genes that overexpress normal or modified UCp , And animals that have lost UCP3 (knockouts) Physiological role and / or activity and / or level and / or structure of UCP3 Manufactured to allow evaluation of the effects of structural changes. Transgenic animals Also screens or tests for substances that modulate UCP3 expression and / or activity Could also be used.   Other advantages of the invention will become apparent on reading the following description.   O.D., incorporated herein by reference. Boss et al., FEBS Lett. 408, 39 (1997) Brief description of the drawings described in: Figure 1: Human UC obtained using the ClusterIW Multiple Sequence Alignment program       P3_L, UCP3_S, UCP2 and alignment of amino acid sequences of UCP (KC Worl       ey, Human Genome Center, Baylor College of Medicine). Array is one character       Shown in notation. Insert into sequence to optimize alignment       Spaces marked are indicated by dashes. Potential transmembrane α-helix is underlined       And number them using Roman numerals (I-VI). Possible pre       The nucleotide binding domain (PNBD) is double underlined. Mitochondria       The three signature domains of the energy transfer protein are boxed. Mitoko       The 14 amino acids that are completely conserved in the dhria carrier are asterisks.       Indicated by FIG. 2: Expression of UCP2 and UCP3 in human and rat tissues. (A) All UCP2 cDNA       including³²Various human tissues hybridized with P-labeled human UCP2 probe       Autoradiogram of Northern blot of poly (A) RNA. Molecular size mer       Kerr is indicated in kb. Insert: UCP2 expression in total RNA. Brown adipose tissue (BAT); white       Adipose tissue (WAT). (B) Contains all UCP3 cDNA³²P-labeled human UCP3 probe       Northern blots of poly (A) RNA from various human tissues hybridized.       Tradogram. Insertion: UCP3 expression in total RNA. Includes all UCP2 cDNA³²P-       Poly (A) RNA of various human tissues hybridized with labeled human UCP2 probe       Autoradiograms of Northern blots. Molecular size markers are shown in kb       You. Insert: UCP2 expression in total RNA. Brown adipose tissue (BAT); white adipose tissue (WAT)       ). (C) Northern blot autoradiograms of various rat tissues.³²       The P-labeled rat atypical UCP cDNA probe was replaced with two rat UCPs, UCP (       (1.4 kb), hybridized with UCP2 (1.7 kb) and UCP3 (2.5 and 2.8 kb)       Let it go.Production of rat atypical UCP cDNA probe and screening of human skeletal muscle cDNA library Leaning (O. Boss et al., FEBS Lett. 408, 39 (1997))   Rabbit tibia ventral muscle and interscapular BAT total RNA were determined by Chomczynski et al. (Anal . Biochem. 162, 156 (1987)). Add 1-2 μg of each RNA to Rigo (dT)_FifteenPrimers and Moloney murine leukemia virus (M-MLV) reverse transcriptase ( Gibco BRL, New York, NY). Briefly, total RNA is 17.5μ 1.5 μl (150 ng) of oligo (dT) in l_FifteenAnd mixed. Add the mixture at 70 ° C for 10 minutes Heated and cooled on ice. After brief centrifugation, the RNA was reverse transcribed for 1 hour at 41 ° C (total l) I did. Aliquots of the first strand cDNA were stored at -20 ° C until use. Polymerase chain reaction was performed using Perkin Elmer DNA Thermal Cycler 480 (Perkin Elmer, Lausanne, Switzerland). First strand cDNA (2 μl) was transferred between rat UCP species. Oligonucleotide primers corresponding to conserved domains: Was amplified with GenBank Accession M11814 in a total volume of 50 μl. Similar size Unique fragments were obtained in BAT and tibialis anterior. The sequence of the BATPCR product is The sequence of the tibialis anterior muscle PCR product was identical to that of rat UCP. Was 60% homologous. This fragment, called rat atypical UCP cDNA, Nucleotide [α-³²Radioactively labeled with [P] dCTP and used as a probe for human skeletal muscle cDN A library was used for screening (Stratagene, # 936215, La Jolla, C A).   About 1 million phages (Human Muscle cDNA) of human skeletal muscle cDNA library Screening using the rat atypical UCP cDNA probe (19) March 8, 1994). Screening led to the isolation of 10 positive clones. Of these, three different categories were classified according to the manufacturer's instructions into plasmids. Purified using the kit for purification (Quiagen, Santa Clarita, CA) Markers using otide primers M13-20 and T3 and others specific for the gene The sequence is determined on both strands with the ABI373A automated sequencer according to the semi-protocol. Until they were sequenced. These three containing 309, 312 and 275 amino acids The predicted peptide of the clone of FIG. 1 is shown in FIG. 1 (O. Boss et al., FEBS Lett. 9 (1997)). The 309 amino acid protein is UCP2 (GenBank Accession U8281). 9) and was isolated by Fleury et al., (Nature Genet. 15, 269 (1997)). ing. The other two proteins share the first 275 amino acids and Indicates that they are isoforms. These isoforms are UCP3 RNA (G. Solanes et al., J. Bio. l. Chem. 272, 25433 (1997)). They are 56% less than human UCP and UCP3, respectively. And 73% homology, and are therefore considered as novel proteins, UCP3 long and short, UCP3_LAnd UCP3_S(GenBank Accession U84763 and And U82818). As with other mitochondrial carriers, UCP2 and UCP3_LHas 6 possible Includes transmissive transmembrane (FIG. 1). Possible purine nucleotide binding domains are , UCP2 from nucleotides 276 to 298, UCP3_LFrom 279 to 301 You. UCP3_SAre involved in the control of six possible transmembrane domains, or uncoupling activities of UCP. No conferring purine nucleotide binding domain (F. Bouillaud et al., EMBO  J. 13, 1990 (1994)).   The binding of guanosine diphosphate (GDP) to UCP is H⁺And Cl^-Ion transmission Induces conformational changes that lead to inhibition of sex (J. Nedergaard et al., New Com prehensive Biochemistry (Bioenergetics) L. Ernster, Ed. (Elsevier Scienc e, Stockholm, 1992), vol. 23, 385). Therefore, UCP3_SDepends on GDP for its bioactivity Appears to work without control. As shown in FIG. 1, the mitochondrial carrier Other properties are UCP2, as well as UCP3_LAnd UCP3_SFound in the first, third and And fourth potential transmembrane domains (O. Boss et al., FEBS Lett. 408, 39 (19 97)), and conserved residues of 14 mitochondrial carriers (F. Palmieri, FEBS Lett. . 346, 48 (1994)).   UCP2 and UCP3 are stronger than UCP compared to other mitochondrial carrier families. (With respect to amino acids and nucleotides) (O. Boss et al., FEB S Lett. 408, 39 (1997)). In fact, its homology with UCP is 55% and 5%, respectively. 6%, whereas the most homologous mitochondrial protein, 2-oxoglu The tallate / maleate carrier is 32%. Therefore, UCP and TCP3 are UCP families. Should belong to Lee. In fact, Fleury et al., (Nature Genet. 15, 269 (1997 )) And Gimeno et al. (Diabetes 46, 900 (1997)) shows that UCP2 is expressed in yeast. Showed mitochondrial uncoupling properties, while Gong et al. (J. Bi ol. Chem. 272, 24129 (1997)) and Boss et al. (J. Biol. Chem. 273, 5, (19 88)) indicates that UCP3 is yeast or C, respectively._TwoC₁₂When expressed in mouse myoblasts, It shows that it has tochondrial uncoupling properties.Comparison of distribution of UCP2 and UCP3 in human and rat tissues (O. Boss et al., F EBS Lett. 408, 39 (1997))   The tissue distribution of UCP3 was compared to UCP2 by Northern blot. For this purpose, The following materials were used: −Human tissue samples   Many human tissue poly (A) RNA membranes (# 7760-1) were purchased from Clontech Laboratories Inc. (P alo Alto, CA). Abdominal white adipose tissue (10 to 40 g) or abdominal skeletal muscle ( 800 mg) of the fragment were obtained during abdominal surgery. Around the kidney weighing about 1.5 g ( perirenal) was obtained during renal surgery in children (mean age: 3 months) . The project consists of the Ethical Committee of the Department of Surgery, Facult y of Medicine, approved by the University of Geneva. −Rat tissue sample   Seven-week-old male Sprague-Dawley rats with free access to standard laboratory chow were Maintained on a 12 hour light / dark cycle. Killed by cervical dislocation. All experiments are , Went according to our established instructions.Northern analysis   Total RNA (10-20 μg) was obtained from Lehrach et al., (Biochemistry 16, 4743 (1977)). Electrophoresis on a 1.2% agarose gel containing formaldehyde as described in And nylon membrane (Electran Nylon Blotting Membrane, BDH Laboratory Supplies , Poole, United Kingdom) with a suction device (Stratagene, La Jolla, CA). About 1 × 10⁹dpm / μg DNA specific activity [α-³²P] dCTP (3000 Ci / mmol) (Ame rsham, Bucks, United Kingdom) random primer (Megaprime DNA Labellin) g System, Amersham, Bucks, United Kingdom) . The RNA membrane was hybridized with QuikHyb (Stratagene, La Jolla, CA) for 2 hours at 65 ° C. And then 2 × SSC (1 × SSC is 150 mM NaCl, 15 mM sodium citrate, p H7.0) /0.1% SDS solution twice at 50 ° C. for 5 minutes, and 0.1 × SSC / 0.1 Washed at 50 ° C. for 5 minutes with% SDS. The membrane was converted to Hyperfilm ECL film (Amersham, Bucks, United Kingdom) at 80 ° C. with intensifying screens. The standard RNA used was And Kb RNA Ladder of Gibco BRL (New York).   As shown in FIG. 2A, a 1.7 kb size UCP2 signal was generated in all the tested tissues. Was detected. UCP2 was expressed at the highest level in BAT> white adipose tissue> skeletal muscle. By comparison, UCP3 (2.3 kb signal) expression is restricted to skeletal muscle and heart I have. In the latter tissue, UCP3 expressed UCP3 with 10 times less intensity than skeletal muscle ( (FIG. 2B). Direct read of Northern blot hybridized with UCP2 and UCP3 probes Close comparisons show that UCP3 is more strongly expressed in skeletal muscle than UCP2. UCP3 length Probes specific to the form or the truncated form give the same signal at 2.3 kb in both forms. Quantitation of the signal intensity indicates that both forms are expressed at similar levels in human skeletal muscle. (O. Boss et al., FEBS Lett. 408, 39 (1997)).   The tissue distribution of UCP2 mRNA and UCP3 mRNA was also determined by the three rat UCPs, UCP and UC. Performed using a rat atypical UCP cDNA probe that hybridizes to P2 and UCP3. (O. Boss et al., FEBS Lett. 408, 39 (1997)). As shown in FIG. P2 is expressed in all tissues studied: heart> BAT> white adipose tissue> skeletal muscle. Hi The major difference from UCP2 is its high expression level in the heart. UCP3 is the best in BAT At a high level, at a high level, the tensor fascia latae (fast twitch, sugar Decomposed), anterior tibia (rapid twitch, oxidative-glycolytic) and gastrocnemius (mixed) It is expressed in soleus muscle (slow twitch, oxidative) at high levels. This is because UCP3 is oxidative bone It shows that it is more strongly expressed in glycolytic rather than skeletal muscle. In rats, UC P3 is also at lower levels, but in the heart and kidneys, and sometimes white adipose tissue Detected in.   As seen in FIG. 2C, the UCP3 signal is a doublet, while its UCP3 Is unique. Messenger size is 1. in UCP compared to RNA ladder. 4 and 1.8 kb, 1.7 kb for UCP2 and 2.5 and 2.8 kb for UCP3.   The results therefore show that the tissue distribution of UCP2 and UCP3 in humans is that of rat UCP Very different: UCP2 expression is ubiquitous and UCP3 is highly specific to skeletal muscle It is a target.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 C07K 14/705 C07K 14/705 C12N 1/21 C12N 1/21 C12P 21/02 C C12P 21 / 02 (C12N 1/21 // (C12N 1/21 C12R 1:19) C12R 1:19) C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, L S, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, M), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW

Claims (1)

[Claims] 1. Encoding the uncoupling protein (U CP3 _L) characterized by the amino acid sequence shown in SEQ ID NO: 4, or comprising a nucleotide sequence shown in SEQ ID NO: 3, or a homologous sequence coding for the same amino acid sequence A DNA fragment, comprising: 2. The DNA fragment according to claim 1, which is derived from human skeletal muscle. 3. It is characterized in that it comprises the nucleotide sequence shown in SEQ ID NO: 5, which encodes the uncoupling protein (UCP3 _S ) having the amino acid sequence shown in SEQ ID NO: 6, or contains the homologous sequence encoding the same amino acid sequence Characteristic, DNA fragment. 4. The DNA fragment according to claim 3, which is derived from human skeletal muscle. 5. An uncoupling protein comprising the amino acid sequence of SEQ ID NO: 4. 6. An uncoupled protein comprising the amino acid sequence of SEQ ID NO: 6. 7. A recombinant DNA fragment comprising the DNA sequence according to any one of claims 1, 2, 3 or 4, or one of its homologous sequences. 8. A DNA molecule comprising a cloning vector into which the DNA sequence according to any one of claims 1 to 4 or 7 has been inserted. 9. 9. The recombinant DNA molecule according to claim 8, wherein the cloning vector is a plasmid or a phage. 10. Comprises a nucleotide according to SEQ ID NO: 3, the vector pBluescript SK ^- are inserted into a recombinant DNA molecule of claim 9, wherein. 11. The recombinant DNA molecule according to claim 10, which has been deposited with the ATCC (number 97999). 12. Comprises a nucleotide according to SEQ ID NO: 5, the vector pBluescript SK ^- are inserted into a recombinant DNA molecule of claim 9, wherein. 13. 13. The recombinant DNA molecule according to claim 12, which has been deposited with the ATCC (No. 209000). 14． A microorganism selected from bacteria, yeast and mammalian cells, comprising the recombinant DNA molecule according to any one of claims 7 to 13. 15. The microorganism according to claim 14, which is an XL-1 Blue MRF 'bacterium (E. coli). 16. A pharmaceutical preparation for correcting UCP3 (UCP3 _L , UCP3 _S ) deficiency by gene therapy, comprising the gene of SEQ ID NO: 3 or 5 and a suitable pharmaceutical vehicle. 17． 17. The pharmaceutical preparation according to claim 16, characterized in that said gene is included in a vector selected from adenovirus, retrovirus, adeno-associated virus, herpes virus, liposome or DNA plasmid. 18. Characterized in that it comprises an antisense oligonucleotide about fragments of the sequence of UCP3 _L and UCP3 _S as active agent, an excess of a pharmaceutical formulation for the correction of _{UCP3 (UCP3 L, UCP3 S)} .